Interactions between molecules is a central theme in living systems. These interactions are key to myriad biochemical and signal transduction pathways. Messages from outside a cell travel along signal transduction pathways into the cell's nucleus, where they trigger key cellular functions. Such pathways in turn dictate the status of the overall system. Slight changes or abnormalities in the interactions between biomolecules can effect the biochemical and signal transduction pathways, resulting in inappropriate development, cancer, a variety of disease states, and even cell senescence and death. On the other hand, it can be extremely beneficial to develop reagents and effectors that can inhibit, stimulate, or otherwise effect specific types of molecular interactions in biochemical systems; including biochemical and signal transduction pathways. Reagents and effectors that effect nucleus interactions may often become very powerful drugs which can be used to treat a variety of conditions.
Current Technology
Several recent studies have shown that a scanning probe microscope “SPM” may be used to study molecular interactions by making a number of measurements. The SPM measurements may include changes in height, friction, phase, frequency, amplitude, and elasticity. The SPM probe can even perform direct measurements of the forces present between molecules situated on the SPM probe and molecules immobilized on a surface. For example, see Lee, G. U., L. A. Chrisey, and R. J. Colton, Direct Measurement of the Forces Between Complementary Strands of DNA. Science, 1994. 266: p. 771-773; Hinterdorfer, P., W. Baumgartner, H. J. Gruber, and H. Schindler, Detection and Localization of Individual Antibody-antigen Recognition Events by Atomic Force Microscopy, Proc. Natl. Acad. Sci., 1996. 93: p. 3477-3481; Dammer, U., O. Popescu, P. Wagner, D. Anselmetti, H. -J. Guntherodt, and G. N. Misevic, Binding Strength Between Cell Adhesion Poteoglycans Measured by Atomic Force Microscopy. Science, 1995. 267: p. 1173-1175; Jones, v. et al. Microminiaturized Immunoassays Using Atomic Force Microscopy and Compositionally Patterned Antigen Arrays, Analy. Chem., 1998 70(7): p. 1233-1241; and Rief, M., F. Oesterhelt, B. Heymann, and H. E. Gaub, Single Molecule Force Spectroscopy on Polysaccharides by Atomic Force Microscopy, Science, 1997. 275: p. 1295-1297. The above studies illustrate that it is possible to readily and directly measure the interaction between and within virtually all types of molecules by utilizing an SPM. Furthermore, recent studies have shown that it is possible to use direct force measurement to detect changes in molecular complex formation caused by the addition of a soluble molecular species. A direct force measurement may elucidate the effect of soluble molecular species on the interaction between a molecular species on an SPM probe and a surface.
Molecular Arrays
The ability to measure molecular events in patterned arrays is an emerging technology. The deposition material can be deposited on a solitary spot or in a variety of sizes and patterns on the surface. The arrays can be used to discover new compounds which may interact in a characterizable way with the deposited material. Arrays provide a large number of different test sites in a relatively small area. To form an array, one must be able to define a particular site at which a deposition sample can be placed in a defined and reproducible manner.
There are four approaches for building conventional molecular arrays known in the art. These prior art methods include 1) mechanical deposition, 2) in situ photochemical synthesis, 3) “ink jet” printing, and 4) electronically driven deposition. The size of the deposition spot (or “domain”) is of particular importance when utilizing an SPM to scan for molecular recognition events. Current SPM technology only allows a scan in a defined area. Placing more domains in this defined area allows for a wider variety of molecular interaction events to be simultaneously tested.
Mechanical deposition is commonly carried out using a “pin tool” device. Typically the pin tool is a metal or similar cylindrical shaft that may be split at the end to facilitate capillary take up of liquid. Typically the pin is dipped in the source and moved to the deposition location and touched to the surface to transfer material to that domain. In one design the pin tool is loaded by passing through a circular ring that contains a film of the desired sample held in the ring by surface tension. The pin tool is washed and this process repeated. Currently, pin tool approaches are limited to spot sizes of 25 to 100 microns or larger. The spot size puts a constraint on the maximum density for the molecular deposition sites constructed in this manner. A need exists for a method that allows for molecular domains of smaller dimensions to be deposited.
In situ photochemical procedures allow for the construction of arrays of molecular species at spatial addresses in the 1-10 micron size range and larger. In situ photochemical construction can be carried out by shining a light through a mask. Photochemical synthesis occurs only at those locations receiving the light. By changing the mask at each step, a variety of chemical reactions at specific addresses can be carried out. The photochemical approach is usually used for the synthesis of a nucleic acid or a peptide array. A significant limitation of this approach is that the size of the synthetic products is constrained by the coupling efficiency at each step. Practically, this results in appreciable synthesis of only a relatively short peptide and nucleic acid specimen. In addition, it becomes increasingly improbable that a molecule will fold into a biologically relevant higher order architecture as the synthetic species becomes larger. A need exists for an alternative method for deposition of macromolecular species that will preserve the molecular formation of interest in addition to avoiding the cost of constructing the multiple masks used in this method.
Ink jet printing is an alternative method for constructing a molecular array. Ink jet printing of molecular species produces spots in the 100 micron range. This approach is only useful for printing a relatively small number of species because of the need for extensive cleaning between printing events. A key issue with ink jet printing is maintenance of the structural/functional integrity of the sample being printed. The ejection rate of the material from the printer results in shear forces that may significantly compromise sample integrity. A need exists for a method that will retain the initial structure and functional aspects of the deposition material and that will form smaller spots than are possible with the above ink jet method.
Electronic deposition is yet another method known for the construction of molecular arrays. Electronic deposition may be accomplished by the independent charging of conductive pads, causing local electrochemical events which lead to the sample deposition. This approach has been used for deposition of DNA samples by drawing the DNA to specific addresses and holding them in a capture matrix above the address. The electronic nature of the address can be used to manipulate samples at that location, for example, to locally denature DNA samples. A disadvantage of this approach is that the address density and size is limited by the dimensions of the electronic array.
A need exists for a molecular deposition technique that will allow for smaller deposition spots (domains). Smaller deposition domains allow for an array to be constructed with a greater density of domains. More domains further allow for a wider variety in the deposition material to be placed on the same array, allowing a user to search for more molecular interaction events simultaneously.
A further need exists for the ability to place these spots at a defined spatial address. Placing the domains at defined spatial addresses allows the user to know exactly what deposition material the SPM is scanning at any given time.
Furthermore, a need exists for a method to make deposition domains with large molecular weight samples that also retains the desired chemical formation. Finally, a need exists for the efficient construction of these molecule domains into an array.
Molecular Detection
All of the above examples are further limited because they require some type of labeling of the deposition sample for testing. Typical labeling schemes may include fluorescent or other tags coupled to a probe molecule. In a typical molecular event experiment, an array of known samples, for example DNA sequences, will be incubated with a solution containing a fluorescent indicator. In the DNA example this would be fluorescently or otherwise labeled nucleic acids, most often a single stranded DNA of an unknown sequence. Specific sequence elements are identified in the DNA sample by virtue of the hybridization of the label to addresses containing known sequence elements. This process has been used to screen entire ensembles of expressed genes in a given population of cells at a particular time or under a particular set of conditions. Other labeling procedures have also been employed, including RF (radio frequency) labels and magnetic labels. These methods are less frequently used, however, than the fluorescent label methods desired above. All of these labels hinder experiments with extra steps, reagents, and in some cases, risk.
Other methods for the detection of the interactions of molecules on a molecular array include inverse cyclic voltametry, capacitance or other electronic changes, radioactivity (such as with isotopes of phosphorous), and chemical reactions. In virtually all cases, some form of labeling of the probe molecule that is added to the array is required. This is a significant limitation of current arrays. A need exists for a method that does not require this extra labeling step.
Scanning Probe Microscopy
A wide variety of SPM instruments are capable of detecting optical, electronic, conductive, and other properties. One form of SPM, the atomic force microscope (AFM), is an ultra-sensitive force transduction system. In the AFM, a sharp tip is situated at the end of a flexible cantilever and scanned over a sample surface. While scanning, the cantilever is deflected by the net sum of the attractive and repulsive forces between the tip and sample. If the spring constant of the cantilever is known, the net interaction force can be accurately determined from the deflection of the cantilever. The deflection of the cantilever is usually measured by the reflection of a focused laser beam from the back of the cantilever onto a split photodiode, constituting an “optical lever” or “beam deflection” mechanism. Other methods for the detection of cantilever deflection include interferometry and piezoelectric strain gauges.
The first AFMs recorded only the vertical displacements of the cantilever. More recent methods involve resonating the tip and allowing only transient contact, or in some cases no contact at all, between it and the sample. Plots of tip displacement or resonance changes as it traverses a sample surface are used to generate topographic images. Such images have revealed the three dimensional structure of a wide variety of sample types including material, chemical, and biological specimens. Some examples of the latter include DNA, proteins, chromatin, chromosomes, ion channels, and even living cells.
In addition to its imaging capabilities, the AFM can make extremely fine force measurements. The AFM can directly sense and measure forces in the microNetwon (10−6) to picoNewton (10−12) range. Thus, the AFM can measure forces between molecular pairs, and even within single molecules. Moreover, the AFM can measure a wide variety of other forces and phenomena, such as magnetic fields, thermal gradients and viscoelasticity. This ability can be exploited to map force fields on a sample surface, and reveal with high resolution the location and magnitude of these fields, as in, for example, localizing complexes of interest located on a specific surface.
Direct Force Measurement
To make molecular force measurements, the AFM probe is functionalized with a molecule of interest. This bio- or chemi-active probe is then scanned across the surface of interest. The molecule tethered to the probe interacts with the corresponding molecule or atoms of interest on the surface being studied. The interactions between the molecule functionalized on the probe and the molecules or atoms on the surface create minute forces that can be measured by displacement of the probe. The measurement is typically displayed as a force vs. distance curve (“force curve”).
To generate a force curve, the tip or sample is cycled through motions of vertical extension and retraction. Each cycle brings the tip into contact with the sample, then pulls the tip out of contact. The displacement of the cantilever is zero until the extension motion brings the tip into contact with the surface. Then the tip and sample are physically coupled as the extension continues. The physical coupling is the result of hard surface contact (Van der Waals interactions) between the probe and the surface. This interaction continues for the duration of the extension component of the cycle. When the cycle is reversed and the tip retracted, the physical contact is broken. If there is no attractive interaction between the tip and sample the tip separates from the sample at the same position in space at which they made contact during extension. However, if there is an adhesive interaction between the tip and sample during retraction, the cantilever will bend past its resting position and continue to bend until the restoring force of the cantilever is sufficient to rupture the adhesive force.
In the case of extendable molecular interactions, the distance between the tip and surface at which a rupture is observed corresponds to the extension length of the molecular complex. This information can be used to measure molecular lengths and to measure internal rupture forces within single molecules. In a force curve an adhesive interaction is represented by an “adhesion spike.” Since the spring constant of the probe is known, the adhesive force (the unbinding force) can be precisely determined. Upon careful inspection of a typical adhesion spike, many small quantal unbinding events are frequently seen. The smallest unbinding event that can be evenly divided into the larger events can be interpreted as representing the unbinding force for a single molecular pair.
The spectra produced by these binding events will contain information about the coupling contacts holding the molecules together. Thus, it is possible to interpret the signature generated by a mechanical denaturation experiment with regard to the internal structure of the molecule. An SPM can further utilize height, friction, and elasticity measurements to detect molecular recognition events. Molecular recognition events are when one molecule interacts with another molecule or atom in, for example, an ionic bond, a hydrophobic bond, electrostatic bond, a bridge through a third molecule such as water, or a combination of these methods.
In an alternative approach, the AFM probe is oscillated at or near its resonance frequency to enable the measurement of recognizance parameters, including amplitude, frequency and phase. Changes in the amplitude, phase, and frequency parameters are extremely sensitive to variations in the interaction between the probe and the surface. If the local elasticity or viscosity of the surface changes as a result of a molecular recognition event, there is a shift in one or more of these parameters.
Others have reported using AFMs and STMs for the deposition of materials. One report is from Chad Mirkin (Northwestern University) in which he used an AFM to write nanometer scale molecule features with short alkane chains. Hong, S., J. Zhu, and C. A. Mirkin, Multiple Ink Nanolithography: Toward A Multiple-Pen Nano-Plotter, Science. 1999, p. 523-525. A need exists, however, for a molecular domain deposition method that is not limited to short chain length molecules. A need exists for a method for depositing longer chain length macromolecules that does not change or hinder the formation of the deposited molecule.
A need exists for an improved apparatus and method for utilization in the detection of molecular interaction events. A need exists for a method for the creation of small, sub-micron scale molecular domains at defined spatial addresses. This apparatus should enable the user to test for a variety of different types of events in a spatially and materially efficient manner by facilitating the deposition, exposure, and scanning of molecular domains to detect a resultant molecular interaction event. Furthermore, an apparatus is needed that enables the placement of a large number of molecular domains in a relatively small area.